12-halogenated forskolin derivatives

ABSTRACT

Novel 12-halogenated forskolin derivatives, intermediates and processes for the preparation thereof, and methods for reducing intraocular pressure utilizing compounds or compositions thereof are disclosed.

This is a division of a pending prior application, Ser. No. 522,754,filed May 14, 1990, now U.S. Pat. No. 5,023,344, which is a division ofa prior application, Ser. No. 390,126, filed Aug. 7, 1989, now U.S. Pat.No. 4,978,678, which is a division of a prior application, Ser. No.932,553, filed Nov. 20, 1986, now U.S. Pat. No. 4,871,764.

The present invention relates to 12-halogenated forskolin derivatives ofFormula 1 ##STR1## wherein X is F, Cl, Br and I; R is hydrogen, ##STR2##where R₁ is hydrogen or lower alkyl, ##STR3## where R², R₃ are the sameor different and are H or lower alkyl; the optical and geometric isomersthereof, which are useful for reducing intraocular pressure, alone or incombination with inert adjuvants.

The present invention also relates to compounds of the formula 2,##STR4## wherein X is as previously defined and Z is either ##STR5##where R₄, R₅ are the same or different and are hydrogen or lower alkyl;or the optical and geometric isomers thereof, which are useful asintermediates for the preparation of the 12-halogenated forskolinderivatives of the present invention. As used throughout thespecification and appended claims, the term "alkyl" refers to a straightor branched chain hydrocarbon radical containing no unsaturation, suchas methyl, ethyl, 1-propyl, 2-propyl, 2-methylpropyl, 1-pentyl, 3-hexyl,and the like. The term "alkanoyl" refers to a monovalent substituentwhich consists of an alkyl group linked through a carbonyl group havingits free valence bond from the carbon of the carbonyl group ##STR6##Examples of alkanoyl groups are formyl, acetyl, propionyl,2,2-dimethylacetyl, hexanoyl, and the like. The term "lower" as appliedto any of the aforementioned groups refers to an alkyl group having acarbon skeleton containing up to and including 6 carbon atoms.

In the formulas presented herein the various substituents areillustrated as joined to the nucleus (labdane structure) by one of twonotations: a solid line (₋₋) indicating a substituent which is in theβ-orientation (i.e., above the plane of the molecule) and a broken line(----) indicating a substituent which is in the α-orientation (i.e.,below the plane of the molecule). The formulae have all been drawn toshow the compounds in their absolute stereochemical configuration.Inasmuch as the starting materials having such labdane nucleus arenaturally occurring or are derived from naturally occurring materials,they, as well as the final products, have a labdane nucleus existing inthe single absolute configuration depicted herein. The processes of thepresent invention, however, are intended to apply as well to thesynthesis of labdanes of the racemic series.

In addition to the optical centers of the labdane nucleus, thesubstituents thereon may also contain chiral centers contributing to theoptical properties of the compounds of the present invention andproviding a means for the resolution thereof by conventional methods,for example, by the use of optically active acids. A wavy line ( ˜ )connecting a group to a chiral center indicates that the stereochemistryof the center is unknown, i.e. the group may exist in any of thepossible orientations. The present invention comprehends all opticalisomers and racemic forms of the compounds of the present invention,where such compounds have chiral centers in addition to those of thelabdane nucleus.

The novel 12-halogenated forskolin derivatives of the present inventionare synthesized by the processes illustrated in the Reaction Schemes Aand B, wherein the groups R, R₁ through R₅ are as previously defined.

To prepare a basic labdane 4, a 1α, 9α-dihydroxylabdane 3 where R₆ islower alkanoyl is condensed with a formamide dialkylacetal of theformula 11, ##STR7## where R₈, R₉ and R₁₀ are independently lower alkyl,or R₈ and R₉ taken together with the nitrogen atom to which they areattached form a group of the formula ##STR8## where X is CHR₁₁ whereinR₁₁ is hydrogen, lower alkyl or a group of the formula OR₁₂ where R₁₂ ishydrogen, lower alkyl or a group of the formula COR₁₃ wherein R₁₃ islower alkyl and m is 0 or 1. The condensation is preferably performed inthe absence of an added solvent, excess formamide dialkylacetal servingboth as the reactant and solvent. A dipolar aprotic solvent such asdimethylformamide, dimethylacetamide, hexamethylphosphoramide ordimethylsulfoxide may be employed, however, as the reaction medium. Thetemperature at which the condensation is conducted is not critical. Acondensation temperature within the range of about 25° to about 100° C.is generally employed to assure a reasonable rate of reaction. Atemperature of about 45° to about 65° C. is preferred.

To furnish a basic labdane of formula 5 wherein R₈ and R₉ are as above,a 7 β-alkanoyloxy-6,β-hydroxylabdane 4 is hydrolyzed to the6,β,7β-dihydroxylabdane 5. The hydrolysis is carried out in an aqueousalkanol such as aqueous methanol, ethanol, 1- or 2-propanol ort-butanol, aqueous methanol being preferred, containing an alkalicarbonate such as lithium, sodium or potassium carbonate, potassiumcarbonate being preferred, at a hydrolysis temperature within the rangeof about 10° to about 75° C., a hydrolysis temperature of about 25° C.being preferred.

To introduce a 6,β, 7β-carbonate function into the basic labdanenucleus, i.e., to prepare a compound of formula 6, a 6β,7β-dihydroxylabdane of formula 5 is treated with a compound of formula12. ##STR9## wherein Hal is bromo or chloro, preferably chloro, in thepresence of an organic base such as trimethyl- or triethylamine,pyridine, lutidine or collidine at a reduced temperature within therange of about -25° to about 25° C. The preferred organic base ispyridine and the preferred reaction temperature is about 0° C.

Alternatively labdane 6 can be prepared by treating the 6β, 7β-dihydroxylabdane 5 with a -compound of the formula ##STR10## whereinR₁₄ is imidazole, ##STR11## in the presence of an organic base such astrimethyl or triethylamine, diisopropylethylamine, etc., at atemperature ranging from 25° to 150° C. for a time period of 30 to 120minutes.

Labdane 6 is treated with a silyl halide of the formula 13 ##STR12##where R₁₅, R₁₆, R₁₇ are independently lower alkyl and Hal is a halogen,preferably Cl, in the presence of a base such as potassium hydride,lithium diisopropylamide or, preferably, lithiumbis[trimethylsilyl]amide to form labdane 7. Typically this reaction iscarried out in the presence of an aprotic solvent, e.g. tetrahydrofuran,diethyl ether, dimethoxy ethane, toluene, etc., at a temperature of -25°to 25° C. for a period of time of from 1 to 3 hours.

To introduce a halogen group at the 12 position, labdane 7 is reacted ina conventional manner with a N-halosuccinimide such as N-bromo-,N-chloro-, N-iodosuccinimide, etc., or other standard suitablehalogenating agents such as N-halophthalimide or N-halocaprolactam,under the influence of fluoride ion. Tetramethylammonium fluoride,tetraethylammonium fluoride, benzyltrimethylammonium fluoride andpotassium fluoride dicyclohexyl-18-crown-6 are suitable sources offluoride ion, with tetra-n-butylammonium fluoride being preferred.Typically when using N-halosuccinimide, the reaction is carried out in apolar aprotic solvent, e.g. diethyl ether, tetrahydrofuran, or dimethoxyethane at a temperature of -65° to 0° C. for a time period of 5 to 60minutes to form labdane 8, where Hal is a halogen (Cl, Br, I).

The removal of the 6β,7βcarbonate function is achieved by hydrolyzingcompound 8 by conventional means. Typically compound 8 is treated with abasic material, e.g. K₂ CO₃, Li₂ CO₃, Na₂ CO₃, etc., in the presence ofwater and a miscible organic solvent, e.g. methanol, ethanol, dioxane,tetrahydrofuran, etc., at a temperature ranging from 25° to 60° C. for atime period of 1 to 8 hours in order to form compound 9.

Compound 9 is then subjected to hydrolysis under acidic conditions toform 12-halogenated desacetyl forskolin of the invention of the formula10. Typically this hydrolysis is carried out in the presence of anorganic acid e.g. acetic acid, citric acid, oxalic acid, etc., in waterand a miscible organic solvent, e.g. methanol, ethanol, dioxane,tetrahydrofuran, etc. at a temperature of 0° to 60° C. for a time periodof 1 to 36 hours to obtain compound 10.

In an alternative procedure, (Reaction Scheme B) a dicarbonate labdaneof the formula 14 is selected. Such labdanes are known in the art or canbe synthesized utilizing conventional techniques. In this regard,reference is made to S. V. Bhat et. al., J. Chemical Society 1982, 767.Labdane 14 is then reacted with the silyl halide 13, in the same mannerpreviously described for reaction of compound 6, to form labdane 15having a silyl ether group where R₁₅, R₁₆ and R₁₇ are as previouslydefined.

The introduction of a halogen group at the 12-position of labdane 15 iscarried out in the same manner as previously described for the formationof compound 8 from compound 7 except that the halogen group isrestricted to F and Cl, to form labdane 16, where Hal is F or Cl.

For the case where Hal is F, labdane 14 is treated with a base, aspreviously described for the synthesis of labdane 7, and then allowed toreact with acetyl hypofluorite. Acetyl hypofluorite is generated fromdilute fluorine in nitrogen and sodium acetate, as described by Rozenand Brand, Synthesis 1985, 665.

The resulting dicarbonate 16 is removed by subjecting labdane 16 tobasic hydrolysis. Typically this is carried out, as previouslydescribed, with an inorganic base, e.g. K₂ CO₃, Li₂ CO₃, Na₂ CO₃, etc.,in the presence of water and a miscible solvent, e.g. methanol, ethanol,dioxane, etc., at a temperature ranging from 25° to 60° C. for a timeperiod of 1 to 8 hours in order to form compound 10 where Hal is F orCl.

The dicarbonate can be removed in two steps by subjecting labdane 16 tomild basic hydrolysis. Typically this is carried out with an inorganicbase, e.g. NaHCO₃ or KHCO₃ in the presence of water and a misciblesolvent e.g. methanol, ethanol, dioxane, tetrahydrofuran, etc. at atemperature ranging from 10° to 35° C. for a time period of 6-24 hoursin order to form compound 17 where Hal is F or Cl. Labdane 10 can thenbe obtained from 17 by the conditions described above.

To form compound 1 of the invention where R is ##STR13## compound 10 isreacted under standard acylating conditions. Typically compound 10 isreacted with an acylating agent, e.g. anhydrides, (R₁ C)₂ O, or acidhalides, ##STR14## where Hal is a halogen such as Cl, Br, etc., in thepresence of a basic solvent such as pyridine, collidina, lutidine,triethylamine etc., at a temperature of 0° to 30° C. for a time periodranging from 1 to 24 hours.

To form compound 1 of the invention where R is ##STR15## compound 10 isreacted with tetrahydrofuroic acid, ##STR16## in the presence of1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in an inertsolvent such as methylene chloride, chloroform benzene or toluene at atemperature of 0° to 30° C. for a time period of 1 to 24 hours.

To form compound 1 of the invention where R is ##STR17## compound 10 isreacted with potassium (2,2-dimethyl-1,3-dioxolano-4-yl)oate in thepresence of 1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridinehydrochloride in an inert solvent such as methylene chloride,chloroform, benzene or toluene of a temperature of 0° to 30° C. for atime period of 1 to 24 hours. The resulting intermediate is thensubjected to hydrolysis under acidic conditions, typically in thepresence of an organic acid e.g. acetic acid, citric acid, oxalic acid,etc. in water and a miscible organic solvent e.g., methanol, ethanol,dioxane, tetrahydrofuran, etc. at a temperature of 0° to 60° for a timeperiod of 1 to 72 hours.

To form compound 1 of the invention where R is ##STR18## compound 10 isreacted with an isocyanate of the formula R₂ N═C═O or a carbamoylchloride of the formula ##STR19## in the presence of an organic basesuch as lithium diisopropylamide or, preferably, lithiumbis[trimethylsilyl]amide in a polar aprotic solvent, e.g.tetrahydrofuran, diethyl ether, dimethoxyethane, etc. at a temperatureof 0° to 100° C. for a time period of 1 to 24 hours.

The labdane starting materials for the processes of the presentinvention, i.e., labdanes of formula 3 and 14 wherein R₆ is hydrogen oracyl, are described in U.S. Pat. No. 4,134,986, issued Jan, 16, 1979 toB. S. Bajwa, et. al. or may be prepared from compounds disclosed thereinby conventional processes.

The labdanes of the present invention are useful in the treatment ofelevated intraocular pressure by virtue of their ability to reduceintraocular pressure as determined by the method described by J.Caprioli, et al., Invest. Ophthalmol. Vis. Sci., 25, 268 (1984). Theresults of the determination expressed as percent decrease of outflowpressure is presented in the Table.

                  TABLE                                                           ______________________________________                                                                   DECREASE IN                                                       CONCEN-     OUTFLOW PRES-                                      COMPOUND       TRATION (%) SURE (%)                                           ______________________________________                                        12-chlorodesacetyl                                                                           2           22                                                 forskolin                                                                     12-chloroforskolin                                                                           2           35                                                 hemihydrate                                                                   12-bromodesacetyl                                                                            2           37                                                 forskolin                                                                     12-bromo-7-(2-tetrahydro-                                                                    1           16                                                 furanoyl) desacetyl                                                           forskolin                                                                     12-bromo-7-(2,3-dihydroxy-                                                                   0.25        28                                                 propionyl)-desacetyl                                                          forskolin                                                                     Forskolin (standard)                                                                         0.25        66                                                 ______________________________________                                    

Intraocular pressure reduction is achieved when the present labdanes areadministered to a subject requiring such treatment as an effectivetopical dose of a 0.01 to 3.0% solution or suspension. A particularlyeffective amount is about 3 drops of a 1% preparation per day. It is tobe understood, however, that for any particular subject, specific dosageregimens should be adjusted according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the aforesaid compound. It is to be further understoodthat the dosages set forth herein are exemplary only and that they donot, to any extent, limit the scope or practice of the invention.

Effective amounts of the compounds of the present invention may beadministered to a subject by any one of various methods, for example,orally as in capsules or tablets, parenterally in the form of sterilesolutions or suspensions, in some cases intravenously in the form ofsterile solutions, or suspensions, and topically in the form ofsolutions, suspension or ointments, and by aerosol spray.

Effective quantities of the compounds of the invention may beadministered orally, for example, with an inert diluent or with anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, theaforesaid compounds may be incorporated with excipients and used in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like. These preparations should contain atleast 0.5% of active compound, but may be varied depending upon theparticular form and may conveniently be between 4% to about 70% of theweight of the unit. The amount of active compound in such composition issuch that a suitable dosage will be obtained. Preferred compositions andpreparations according to the present invention are prepared so that anoral dosage unit form contains between 0.1-30 milligrams of the activecompound.

The tablets, pills, capsules, troches and the like may also contain thefollowing ingredients' a binder such as microcrystalline cellulose, gumtragancanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, corn starch and the like; alubricant such as magnesium stearate; a glidant such as colloidalsilicon dioxide; and a sweetening agent such as sucrose or saccharin ora flavoring agent such as peppermint, methyl salicylate, or orangeflavoring may be added. When the dosage unit form is a capsule, it maycontain, in addition to materials of the above type, a liquid carriersuch as a fatty oil. Other dosage unit forms may contain other variousmaterials which modify the physical form of the dosage unit, forexample, as coatings. Thus, tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes, colorings and flavors. Materials used inpreparing these various compositions should be pharmaceutically pure andnon-toxic in the amounts used.

For the purpose of parenteral or topical therapeutic administration, theactive compounds of the invention may be incorporated into a solution,suspension, ointment or cream. These preparations should contain atleast 0.01% of active compound, but may be varied between 0.5 and about5% of the weight thereof. The amount of active compounds in suchcompositions is such that a suitable dosage will be obtained. Preferredcompositions and preparations according to the present invention areprepared so that a parenteral dosage unit contains between 0.01 to 10milligrams of active compound.

The solutions or suspensions for topical or parenteral administrationmay also include the following components: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. The parenteral preparation can be enclosed inampules or disposable syringes; the topical preparation may be enclosedin multiple dose vials or dropping bottles, made of glass or plastic.

The following examples are for illustration purposes only and are not tobe construed as limiting the invention. All temperatures are given indegrees Centigrade.

EXAMPLE 1 (a) Desacetylforskolin 1,9'6,7-Dicarbonate11,12-Enol-t-butyldimethylsilyl ether

Forskolin 1,9:6,7-dicarbonate (1.1846 g, 2.82 mmole) was dissolved in 25ml of dry tetrahydrofuran (THF) and t-butyldimethylsilyl chloride (0.64g, 4.27 mmole) was added, followed by 3.3 ml of 1 M lithiumbis(trimethylsilyl)amide (3.3 mmole). The reaction was complete after 1hour. The reaction mixture was distributed between aqueous NaHCO₃solution and ether and then the organic phase was dried and evaporated.The residue was triturated well with pentane, giving 1.1288 g (75%) ofdesacetylforskolin, 1,9:6,7-dicarbonate 11,12-enol-t-butyldimethylsilylether, mp 175°-177° C.

ANALYSIS:

Calculated for C₂₈ H₄ O₈ Si: 62.89%C, 7.92%H,

Found: 63.18%C, 7.86%H.

(b) 12-Chlorodesacetylforskolin 1,9:6,7-Dicarbonate

Desacetylforskolin 1,9:6,7-dicarbonate 11,12-enol-t-butyldimethylsilylether of Example 1(a) (0.294 g, 0.550 mmole) was dissolved in 10 ml ofdry THF and chilled to -75° C. N-chlorosuccinimide (0.080 g, 0.60 mmole)was added in 2 ml of THF and then 0.60 ml of 1 M tetra-n-butylammoniumfluoride (0.60 mmole) was added dropwise. After 15 minutes the reactionmixture was evaporated and applied directly to a flash column (5% ethylacetate-CH₂ Cl₂) Evaporation of the product-containing fractions gave0.1546 g (62%) of product. Recrystallization from benzene-pentaneyielded 12-chlorodesacetylforskolin 1,9:6,7-dicarbonate, mp 265°(decomp).

ANALYSIS:

Calculated for C₂₂ H₂₈ C10₈ : 58.08%C, 5.98%H,

Found: 57.89%C, 6.00%H.

(c) 12-Chlorodesacetylforskolin

12-Chlorodesacetylforskolin 1,9:6,7-dicarbonate of Example 1(b) (0.1777g, 0.391 mmole) was stirred for 5 hours in 10 ml of K₂ CO₃ /methanol/H₂O. The reaction mixture was distributed between ether and H₂ O and thenthe organic phase was separated and dried. The crude reaction productwas purified by flash chromatography (30% ethyl acetate-hexane) to give0.0665 g of pure product (42%). Recrystallization from CH₂ Cl₂ -pentaneyielded 12-chlorodesacetylforskolin, mp 175°-177° C.

ANALYSIS:

Calculated for C₂₀ H₂₁ C10₆ : 59.62%C, 7.76%H,

Found: 59.47%C, 7.96%H.

EXAMPLE 2 12-Chloroforskolin Hemihydrate

12-Chlorodesacetylforskolin of Example 1(c) (0.140 g, 0.347 mmole) wasdissolved in 1.5 ml of pyridine to which 0.047 ml of acetic anhydride(0.50 mmole) was then added. The reaction mixture was stirred overnight(about 16 hours) and then distributed between H₂ O and ether. Theaqueous was separated and the organic phase was washed twice with H₂ Oand once with 5% aqueous HCl. The organic phase was then dried,evaporated and purified by flash chromatography (5% ethyl acetate CH₂Cl₂) to give 0.0717 g (46%) of 12-chloroforskolin hemihydrate. Thisproduct was combined with the product of another run and recrystallizedtogether from CH₂ Cl₂ -pentane to give analytically pure material, mp224°-226° C.

ANALYSIS:

Calculated for C₂₂ H₂₃ C10₇.0.5H₂ O: 58.20%C, 7.55%H,

Found: 58.14%C, 7.48%H.

EXAMPLE 3 12-Chloro-7-(2-tetrahydrofuranoyl)desacetylforskolin

12-Chlorodesacetylforskolin (0.2700 g, 0.67 mmole) of Example 1(c) wasdissolved in 5 ml of CH₂ Cl₂ and then tetrahydrofuroic acid (0.092 g,0.80 mole), dicyclohexylcarbodiimide (0.144 g, 0.70 mmole) and4-dimethylaminopyridine (0.085 g, 0.70 mmole) were added sequentially.The reaction mixture was stirred overnight (about 16 hours) and thenether was added and the resulting dicyclohexyl urea was filtered off.The residue was purified by flash chromatography (5% ethyl acetate-CH₂Cl₂) and the product-containing fractions were evaporated and theresidue recrystallized from benzene-pentane to give 0.0851 g (25%) of12-chloro-7-(2-tetrahydrofuranoyl) desacetylforskolin.

ANALYSIS:

Calculated for C₂₅ H₃₇ C10₈ : 59.93%C, 7 44%H,

Found: 59.55%C, 7.35%H.

EXAMPLE 4 (a) Forskolin 1,9-dimethylformamide acetal

Forskolin (100 mg) was dissolved in 1 ml of dimethylformamidedimethylacetal. The mixture was stirred 1 hour at room temperature andovernight at 55° under nitrogen. The mixture was dissolved in ether,washed with water, dried over anhydrous sodium sulfate, filtered andconcentrated to an oil. The oil was dissolved in a minimum volume ofdichloromethane and chromatographed using 10 g of silica gel (230-400mesh) Eluant: 8×3 ml of dichloromethane, 8×3 ml of 3%methanol/dichloromethane and 8×3 ml of 5% methanol/dichloromethane.Evaporation of the appropriate fractions followed by drying at 60° C. (1mm) provided 90 mg (79.1%) of forskolin 1,9-dimethylformamide acetal asan oil.

ANALYSIS:

Calculated for C₂₅ H₃₉ NO₇ : 64.49%C, 8.44%H, 3.01%N,

Found: 64.69%C, 8.25%H, 3 09%N.

(b) Desacetylforskolin 1,9-dimethylformamide acetal

A solution of 225 mg of forskolin 1,9-dimethylformamide acetal ofExample 4(a) was stirred at room temperature under nitrogen for 5 hoursin 5 ml of saturated K₂ CO₃ solution in 20% aqueous methanol. Thesolution was diluted with water and extracted twice with ether. Theether extractions were washed twice with water and dried over anhydroussodium sulfate. Filtration followed by evaporation provided an oil whichcrystallized on standing. The crystals were dried at 113° (1 mm) toyield 192 mg, (88.5%) of desacetylforskolin 1,9-dimethylformamideacetal, mp 136°14 144° C.

ANALYSIS:

Calculated for C₂₃ H₃₇ NO₆ : 65.22%C, 8.81%H, 3.31%N,

Found: 65.18%C, 8.76%H, 3 25%N.

(c) Desacetylforskolin 6,7-Carbonate 1,9-Dimethylformamide Acetal

Desacetylforskolin 1,9-dimethylformamide acetal (1.059 g, 2.5 mmole) ofExample 4(b) was refluxed in 25 ml of toluene containing 0.50 g ofN,N'-carbonyldiimidazole (3.08 mmole) and 0.55 ml of triethylamine (4.0mmole). After 3 hours, thin layer chromatography (TLC) showed conversionto one main new product. The reaction mixture was evaporated and applieddirectly to a flash column. Elution with 50% ethyl acetate-hexane gave0.976 g (87%) of product after combination of the appropriate fractions.Analytically pure desacetylforskolin 6,7-carbonate 1,9-dimethylformamideacetal was obtained by recrystallization from hexane, mp 138°-140° C.

ANALYSIS:

Calculated for C₂₄ H₂₅ NO₇ : 64.12%C, 7.85%H, 3.12%N,

Found: 63.91%C, 7.98%H, 3 08%N.

(d) 12-Bromodesacetylforskolin 6,7-Carbonate 1,9-dimethylformamideAcetal

Desacetylforskolin 6,7-carbonate 1,9-dimethylformamide acetal of Example4(c) (0.450 g, 1.0 mmole) was dissolved in 10 ml. of dry THF andt-butyldimethylsilyl chloride (0.20 g, 1.3 mmole) and lM lithiumbis(trimethylsilyl)amide (1.5 ml, 1.5 mmole) were added. After 30minutes at room temperature the reaction was distributed between NaHCO₃solution and the organic phase was separated and dried. Evaporation gavean oil that resisted crystallization, and was used in the next stepwithout further purification. The NMR (CDCl₃) was consistent with theformation of 12-bromodesacetyl forskolin-6,7-carbonate1,9-dimethylformamide 11,12-enol-t-butyldimethylsilyl ether. The silylenol ether was dissolved in 10 ml of dry THF and chilled to -65° C.N-Bromosuccinimide (0.20 g, 1.12 mmole) was then added, followed by 1.1ml of 1 M tetra-n-butylammonium fluoride (1.1 mmole). After 15 minutesthe reaction mixture was evaporated and applied directly to a silica gelcolumn. Elution with 5% ethyl acetate-CH.sub. 2 Cl₂ gave 0.3718 g (70%for two steps) of product after evaporation of the appropriatefractions. Analytically pure 12-bromodesacetylforskolin 6,7-carbonate1,9-dimethylformamide acetal was obtained by recrystallization frombenzene-pentane, mp 157°-159° C.

ANALYSIS:

Calculated for C₂₄ H₃₄ BrNO₇ : 54.55%C, 6.49%H, 2.65%N,

Found: 54.35%C, 6.50%H, (2.41%N.

(e) 12-Bromodesacetylforskolin 1,9-dimethylformamide Acetal

12-Bromodesacetylforskolin 6,7-carbonate 1,9-dimethylformamide acetal ofExample 4(d) (0.2831 g, 0.536 mmole) was stirred for 2 hours in 5 ml ofmethanol/H₂ O/ K₂ CO₃. At the end of this time the reaction mixture waspoured into H₂ O and the pH was carefully adjusted to 6-7. The productwas extracted into CH₂ Cl₂, evaporated to an oil and purified by flashchromatography (10% ethyl acetate-CH₂ Cl₂) Evaporation of theappropriate fractions gave 0.1885 g of a foam (70%) of12-bromodesacetylforskolin 1,9-dimethylformamide acetal, mp 85°-87° C.

ANALYSIS:

Calculated for C₂₃ H₃₆ BrNO₆ : 54.98%C, [7.22%H, 2 79%N,

Found: 55.05%C, 7.42%H, 2.55%N.

(f) 12-Bromodesacetylforskolin

12-Bromodesacetylforskolin 1,9-dimethylformamide acetal of Example 4(e)(0.5187 g, 1.03 mmole) was stirred overnight (about 16 hours) in 25 mlof 1:1 methanol-80% acetic acid. The reaction mixture was thenevaporated and the residue distributed between ether and NaHCO₃solution. The organic phase was separated, dried, and evaporated andthen purified by flash chromatography (30% ethyl acetate-CH₂ Cl₂) togive, after evaporating the appropriate fractions, 0.4441 g (96%) ofchromatographically pure product Recrystallization from CH₂ Cl₂ yieldedanalytically pure 12-bromodesacetylforskolin, mp 133°-135° C. (decomp).

ANALYSIS:

Calculated for C₂₀ H₃₁ BrO₆ : 53.69%C, 6.99%H,

Found: 53.80%C, 7.11%H.

EXAMPLE 5 12-Bromoforskolin

12-Bromodesacetylforskolin (0.2448 g, 0.547 mmole) of Example 4(f) wasdissolved in 5 ml of pyridine and then 0.075 ml of acetic anhydride(0.79 mmole) was added. The reaction mixture was stirred for 3 days andthen evaporated and distributed between ether and 5% HCl. The organiclayer was separated, dried, evaporated and purified by flashchromatography (10% ethyl acetate CH₂ Cl₂) to give 0.1475 g (55%) ofproduct. Analytically pure 12-bromodesacetylforskolin was obtained byrecrystallization from benzene-pentane, mp 203° (d).

ANALYSIS:

Calculated for C₂₂ H₃₃ BrO₇ : 53.99%C, 6.80%H,

Found: 53.86%C, 6.82%H.

EXAMPLE 6 12-Bromo-7-(2-tetrahydrofuranoyl)desacetyl forskolin

12-Bromodesacetyl forskolin (0.2678 g, 0.60 mmole) of Example 4(f) wasdissolved in 5 ml CH₂ Cl₂ containing 0.120 g (1.03 mmole) oftetrahydrofuroic acid. Dicyclohexylcarbodiimide was added (0.144 g, 0.70mmole), followed by 4-dimethylaminopyridine (0.085 g, 0.70 mmole) andthe reaction mixture was allowed to stir overnight. It was then dilutedwith ether and filtered through celite. Evaporation gave a gummy residuethat was purified by flash chromatography (10% ethyl acetate-CH₂ Cl₂) togive a mixture of diastereomers, after combining and evaporating theappropriate fractions. Recrystallization from benzene-pentane gave 0.136g of analytically pure 12-bromo-7-(tetrahydrofurano-2-yl)desacetylforskolin (42%), mp 203° C. (d).

ANALYSIS:

Calculated for C₂₅ H₃₇ BrO₈ : 55.04%C, 6.84% H,

Found: 54.93%C, 6.81%H.

EXAMPLE 7 12-Bromo-7-(2,2-dimethyl-1,3-dioxolano-4-yl)desacetylforskolin

12-Bromodesacetylforskolin (0.680 g, 1.52 mmole) of Example 4(f) wasdissolved in 10 ml of CH₂ Cl₂. To the solution was added sequentiallypotassium (2,2-dimethyl-1,3-dioxolano-4-yl)oate (0.313 g, 1.70 mmole),4-dimethylaminopyridine hydrochloride (0.270 g, 1.70 mmole) anddicyclohexylcarbodinide (0.351 g, 1.70 mmole). The reaction mixture wasstirred overnight and then ether was added and the precipitated ureafiltered off. The filtrate was evaporated and the residue purified byflash chromatography (10% ethyl acetate-CH₂ Cl₂) to give, afterevaporation of the appropriate fractions and scrupulous drying (80° C.,0.1 mm Hg), 0.4217 g (48.9%) of analytically pure12-bromo-7-(2,2-dimethyl-1,3-dioxolano-4-yl)desacetylforskolin, m p.115°-120° C.

ANALYSIS:

Calculated for C₂₆ H₃₉ BrO₉ : 54.26%C, 6.83%H,

Found: 54.25%C, 6.85%H.

EXAMPLE 8 12-Chlorodesacetylforskolin 1,9-dimethylformamide acetal

Desacetylforskolin 6,7-carbonate 1,9-dimethylformamide acetal of Example4(c) (1.80 g, 4.0 mmole) was dissolved in 40 ml of dry THF to which wasthen added 0.80 g of t-butyldimethylsilyl chloride (5.31 mmole) followedby 6.0 ml of 1 M lithium bis(trimethylsilyl)amide in hexanes. Thisreaction mixture was stirred for 1 hour at which time TLC showed goodconversion to the corresponding enol silyl ether. The reaction mixturewas then distributed between ether and NaHCO₃ solution, after which theorganic phase was separated, dried and evaporated. The residual oil wasdissolved in 30 ml of dry THF which was then chilled to -65° C.N-chlorosuccinimide was then added (0.60 g, 4.50 mmole), followed by 5.0ml of lM tetra-n-butylammonium fluoride in THF. After 30 minutes at thistemperature the reaction mixture was evaporated and applied directly toa column of 230-400 mesh silica gel, and eluting with 10% ethylacetate-CH₂ Cl₂. The product containing fractions were evaporated andshown to contain an unseparated impurity by NMR, so this material wastaken on to the next step without further purification. It washydrolyzed in 10 ml of K₂ CO₃ /methanol/H₂ O for 4 hours and then thereaction mixture was distributed between H₂ O and ether. Evaporation andpurification by flash chromatography (10% ethyl acetate-CH₂ Cl₂) gave0.4825 g of 12-chlorodesacetylforskolin 1,9-dimethylformamide acetal,m.p. 80°-85° C., after rigorous removal of the solvents at 0.1 mm Hg(26% for the three steps).

ANALYSIS:

Calculated for C₂₃ H₃₆ ClNO₆ : 60.31%C, 7.92%H, 3.06%N,

Found: 60.61%C, 7.99%H, 2 68%N.

It is anticipated that the resultant 12-chlorodesacetylforskolin1,9-dimethylformamide acetal can be employed, using analogous proceduresto those of Example 4(f), to obtain 12-chlorodesacetylforskolin.

EXAMPLE 9 12-Chlorodesacetylforskolin 6,7-Carbonate

12-Chlorodesacetylforskolin 1,9:6,7-dicarbonate of Example 1(b) (0.210g, 0.461 mmole) was dissolved in 4 ml of THF and then 2 ml of saturatedaqueous NaHCO₃ solution was added. The reaction mixture was stirredovernight (about 16 hours) and then distributed between ether and H₂ O.The organic phase was dried and evaporated and then purified by flashchromatography (5% ethyl acetate-CH₂ Cl₂) Evaporation of the productcontaining fractions gave 0.143 g (72%) of product. Recrystallizationfrom benzene-pentane yielded 12-chlorodesacetylforskolin 6,7-carbonate(analytically pure).

ANALYSIS:

Calculated for C₂₁ H₂₉ C10₇ : 58.80%C, 6.82%H,

Found: 58.61%C, 6.83%H.

It is anticipated that the resultant 12-chloroforskolin 6,7-carbonatecan be employed, using a procedure analogous to that of Example 1(c) toobtain 12-chloro desacetylforskolin.

EXAMPLE 10 12-Bromo-7-(2,3-dihydroxypropionyl)desacetylforskolin(Diastereomer A)

The acetonide (0.350 g, 0.608 mmole) of Example 7 was stirred for twodays in a solution composed of 5 ml of 80% aqueous acetic acid and 1 mlof methanol. At the end of this time the reaction mixture wasdistributed between ether and H₂ O, and then the organic phase waswashed with NaHCO₃ solution. The residue that remained upon evaporationof the organic phase was purified by flash chromatography (50% ethylacetate-hexane) to give unreacted starting material and product as amixture of diastereomers. The recovered starting material wasresubjected to the conditions of the hydrolysis for an additional threedays and then worked up as before. Flash chromatography of the combinedproducts from the two runs separated the diastereomers when ether wasused as an eluent. The fractions containing the fast running isomer(diastereomer A) were combined, evaporated and crystallized fromether-pentane to give 0.0937 g (29%) of12-bromo-7-(2,3-dihydroxypropionyl)desacetylforskolin (diastereomer A)mp 185°-187° C.

ANALYSIS

Calculated for C₂₃ H₃₅ BrO₉ : 51.59%C, 6.59%H,

Found: 51.78%C, 6.63%H.

EXAMPLE 11 12-Bromo-7-(2,3-dihydroxypropionyl)desacetylforskolin(Diastereomer B)

The acetonide (0.350 g, 0.608 mmole) of Example 7 was stirred for twodays in a solution composed of 5 ml of 80% aqueous acetic acid and 1 mlof methanol. At the end of this time the reaction mixture wasdistributed between ether and H₂ O, and then the organic phase waswashed with NaHCO₃ solution. The residue that remained upon evaporationof the organic phase was purified by flash chromatography (50%ethylacetate-hexane) to give unreacted starting material and product asa mixture of diastereomers. The recovered starting material wasresubjected to the conditions of the hydrolysis for an additional threedays and then worked up as before. Flash chromatography of the combinedproducts from the two runs separated the diastereomers when ether wasused as eluent. The fractions containing the slow running isomer(diastereomer B) were combined, evaporated and crystallized frombenzene-pentane to give 0.0720 g (22%) of12-bromo-7-(2,3-dihydroxypropionyl)desacetylforskolin (diastereomer B)mp 127°-130° C.

ANALYSIS:

Calculated for C₂₃ H₃₅ BrO₉ : 51.59%C, 6.59%H,

Found: 51 31%C, 6.66%H.

EXAMPLE 12 (a) 12-Fluorodesacetylforskolin 6,7-carbonate

Forskolin 1,9'6,7-dicarbonate (1.60 g, 3.80 mmole) was dissolved in 45ml. of dry THF and then 4.5 mL of 1 M lithium bis[trimethylsilyl]amidewas added. The reaction mixture was stirred at room temperature for 30minutes and then the solvent was removed under reduced pressure and theresidue re-dissolved in 40 mL of dry THF. This solution of the lithiumenolate was added rapidly to a solution of acetyl hypofluorite, preparedin the following manner: To 8.2 g of sodium acetate (0.1 mole) was added6.0 g of glacial acetic acid (0.1 mole). The slurry was mixed well witha spatula and allowed to stand with occasional stirring until a uniform,free-flowing powder was obtained. This was then suspended in CFCl₃ (450mL) at -70° C. and a mixture of fluorine in nitrogen (10%) wasintroduced slowly through a fritted glass inlet. After 30 minutes,addition of an aliquot to acidic aqueous KI indicated an oxidizingsolution. After the addition of the lithium enolate, the solution wasstirred for 5 minutes in the cold and then quenched with 400 mL of 5%sodium thiosulfate solution. The aqueous phase was separated and theorganic phase was washed further with NaHCO₃ solution and then water.After drying and evaporating the organic phase the residue was treatedfor 90 minutes with a 1:1 solution of saturated NaHCO₃ solution and THF,to give conversion to the more easily separable 6,7-monocarbonate. Thereaction mixture was distributed between ether and H₂ O, and then theresidue obtained from the dried and evaporated organic phase waspurified by flash chromatography (3% ethyl acetate-CH₂ Cl₂) Combinationof the product containing fractions gave 0.2109 g (13%) of product.Recrystallization from ether-pentane, yielded12-fluorodesacetylforskolin 6,7-carbonate, m.p. 223°-225° C.

ANALYSIS:

Calculated for C₂₁ H₂₉ FO₇ : 61.16%C, 7.09%H,

Found: 61.21%C, 7.25%H.

(b) 12-Fluorodesacetylforskolin

12-Fluorodesacetylforskolin 6,7-carbonate of Example 12(a) (0.190 g,0.46 mmole) was stirred for 1 hour in 2 mL of K₂ CO₃ /methanol/H₂ O. Atthe end of this time it was distributed between CH₂ Cl₂ and H₂ O andthen the residue obtained from the organic phase was purified by flashchromatography (25% ethyl acetate-CH₂ Cl₂). The product containingfractions were combined and recrystallized from methanol-H₂ O to give0.061 g (34%) of 12-fluorodesacetylforskolin, mp 183°-184° C.

ANALYSIS:

Calculated for C₂₀ H₂₁ FO₆ : 62.16%C, 8.09%H,

Found: 61.96%C, 7.94%H.

EXAMPLE 13 12-Fluoroforskolin

12-fluorodesacetylforskolin of Example 12(b) (0.3257 g, 0.843 mmole) wasdissolved in 5 mL of pyridine and then acetic anhydride (0.125 mL, 0.135g, 1.32 mmole) was added. The reacting mixture was stirred for 48 hoursand then distributed between ether and H₂ O. The aqueous phase wasseparated and the organic phase was washed once with H₂ O and once with5% HCl. The residue obtained from the dried and evaporated organic phasewas purified by flash chromatography (5% ethyl acetate-CH₂ Cl₂).Combination and evaporation of the product containing fractions gave0.1272 g (35%) of product. Recrystallization from CH₂ Cl₂ -pentaneyielded 12-fluoroforskolin, m.p. 263°-265° C.

ANALYSIS:

Calculated for C₂₂ H₃₃ FO₇ : 61.66%C, 7.76%H,

Found: 61.46%C, 7.79%H.

EXAMPLE 14 (a) 12-Fluorodesacetylforskolin 1,9-dimethylformamide acetal

12-fluorodesacetylforskolin 6,7-carbonate of Example 12(a) (0.6973 g,1.68 mmole) was stirred for 2 hours at 55° C. in excessdimethylformamide dimethyl acetal. At the end of this time the volatileswere removed under reduced pressure and the residue stirred inmethanolic K₂ CO₃. After one hour the reaction mixture was distributedbetween CH₂ Cl₂ and H₂ O and then the organic phase was dried,evaporated and purified by flash chromatography (30% ethylacetate-hexane). Combination of the product-containing fractions gave0.342 g (46%) of product. Recrystallization from methanol-H₂ O yielded12-fluorodesacetylforskolin 1,9-dimethylformamide acetal, mp 179°-181°C.

ANALYSIS:

Calculated for C₂₃ H₃₆ FNO₆ : 62.56%C, 8.22%H, 3.17%N,

Found: 62.69%C, 8.34%H, 3.05%N.

(b) 12-fluoro-7-(N-methylaminocarbonyl)desacetylforskolin1,9-dimethylformamide acetal

12-Fluorodesacetylforskolin 1,9-dimethylformamide acetal of Example14(a) (0.3418 g, 0.774 mmole) was dissolved in 30 mL of dry THF to whichwas then added 0.077 mL of 1 M lithium bis(trimethylsilyl)amide (0.077mmole) and 0.047 mL (0.046 g, 0.80 mmole) of methyl isocyanate. Thereaction mixture was refluxed for 2 hours and then quenched by theaddition of 0.20 mL H₂ O. Evaporation gave a residue that waschromatographed (50% ethyl acetate-hexane) to give, after combinationand evaporation of the appropriate fractions, 0.230 g (60%) of product.Recrystallization from ether-pentane yielded mp 204°-205° C.

ANALYSIS:

Calculated for C₂₅ H₃₉ FN₂ O₇ : 60.22%C, 7.88%H, 5 62%N,

Found: 60.14%C, 8.29%H, 5.72%N.

(c) 12-fluoro-7-(N-methylaminocarbonyl)desacetylforskolin

12-fluoro-7-(N-methylaminocarbonyldesacetylforskolin1,9-dimethylformamide acetal of Example 14(b), (0.1874 g, 0.376 mmole)was stirred overnight in 10 mL of 1:1 80% aqueous acetic acid:methanol.At the end of this time the reaction was distributed between H₂ O andCH₂ Cl₂ and the organic phase was separated and washed with NaHCO₃solution. The residue obtained from the evaporation of the organic phasewas purified by flash chromatography (20% ethyl acetate-CH₂ Cl₂). Theproduct-containing fractions were combined and recrystallized fromether-pentane and then from methanol-H₂ O to give 0.0831 g of12-fluoro-7-(N-methylaminocarbonyl)desacetylforskolin (49.7%), mp149°-150° C.

ANALYSIS:

Calculated for C₂₂ H₃₄ FNO₇ : 59.58%C, 7.73%H, 3.16%N,

Found: 59.82%, 7.86%H. 2.69%N. ##STR20##

I claim:
 1. A compound of the formulawherein X is F, Cl, Br or I.